Amino acid backbone ionizable lipid, and preparation method and application thereof

Abstract

The application provides an amino acid skeleton ionizable lipid and a preparation method and application thereof, the amino acid skeleton ionizable lipid is modified with amino acid as a core, has more ester groups and peptide bonds, can be rapidly hydrolyzed by enzymes after effectively releasing RNA in the body; the ionizable lipid has four tail structures, can increase the cross-sectional area of the lipid tail, help RNA and other drugs escape from endosomes, and further enhance the transfection effect; the charge of the ionizable lipid is electrically neutral under physiological conditions, reduces the cytotoxicity caused by too many positive charges, further increases the stability of the lipid nanoparticle, and helps to prolong the circulation time of the loaded nucleic acid drug, improve the pharmacokinetic characteristics. The LNP prepared from the ionizable lipid provided by the application, auxiliary phospholipid, cholesterol and PEG lipid has more excellent nucleic acid carrier performance, and can effectively deliver siRNA, mRNA, pDNA and other nucleic acid drugs into cells to play a role.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically relating to an ionizable lipid with an amino acid backbone, its preparation method, and its application. Background Technology

[0002] Cancer vaccines generally fall into four categories: tumor or immune cell vaccines, peptide vaccines, viral vector vaccines, and nucleic acid vaccines. Nucleic acid-based vaccines (DNA or RNA vaccines) are a promising candidate. RNA therapy primarily includes antisense oligonucleotides (ASOs), small interfering RNA (siRNA), small molecular RNA (miRNA), messenger RNA (mRNA), and circular RNA (circRNA), showing great promise in treating a wide range of diseases by manipulating different modes of action. First, nucleic acid vaccines can simultaneously deliver multiple antigens, such as tumor-associated antigens (TAAs) or somatic tumor mutations, triggering humoral and cellular immunity and reducing vaccine resistance. Second, unlike peptide vaccines, nucleic acid vaccines allow APCs to simultaneously or cross-present multiple epitopes of class I and class II patient-specific human leukocyte antigens (HLA types), thus being less restricted by human HLA types and more likely to stimulate a broader T-cell response. However, due to the inherent negative charge and instability of RNA molecules, RNA struggles to overcome biological barriers and reach the cytoplasm. To overcome this challenge, RNA requires a safe, efficient, and stable delivery system to protect nucleic acids from degradation and accelerate cellular uptake and efficient RNA release. Currently, lipid nanoparticles (LNPs) are widely used in drug delivery, but RNA vaccines still require more efficient delivery systems.

[0003] Nucleic acid delivery systems can be broadly categorized into two types: viral vectors and non-viral vectors. Viral vectors offer relatively high transfection efficiency but suffer from drawbacks such as poor safety and targeting. Over the past few decades, liposomes, as a representative of non-viral vectors, have experienced rapid development. A novel type of lipid—ionizable lipid—has been developed. This lipid can be protonated at a weakly acidic pH, making it positively charged, while remaining neutral at physiological pH. The pH sensitivity of ionizable lipids is beneficial for in vivo mRNA delivery because neutral lipids interact less with the anion exchange membranes of blood cells, thus improving the biocompatibility of nanoparticles. When lipid nanoparticles are within the endosomes at a weakly acidic pH, the ionizable lipids acquire a charge, promoting membrane instability and increasing endosome escape from the nanoparticles. However, ionizable lipids still have the following aspects: (i) toxicity issues: ionizable lipids are key components in lipid nanoparticles (LNPs) that trigger acute immune responses and long-term toxicity, but further optimization of lipid structure is still needed; (ii) synthetic complexity: the current synthesis process of ionizable lipids is cumbersome, requiring multiple reaction steps and complex purification steps, which limits large-scale production and rapid screening; (iii) low delivery efficiency: low endosome escape is a common delivery efficiency problem of lipid nanoparticles, and further research is needed to optimize the endosome escape mechanism. Summary of the Invention

[0004] To overcome the problems existing in the prior art, one objective of the present invention is to provide an amino acid-based ionizable lipid. A second objective of the present invention is to provide a method for preparing an amino acid-based ionizable lipid. A third objective of the present invention is to provide a lipid composition. A fourth objective of the present invention is to provide the application of the above-mentioned amino acid-based ionizable lipid and lipid composition in a delivery carrier. A fifth objective of the present invention is to provide a pharmaceutical composition. A sixth objective of the present invention is to provide the application of the above-mentioned amino acid-based ionizable lipid and pharmaceutical composition in the preparation of a drug.

[0005] This invention proposes an ionizable lipid with an amino acid backbone. The lipid's chemical structure comprises three components: (i) an ionizable head group, (ii) a linker group, and (iii) a hydrophobic tail. The head group, a tertiary or secondary amine group, gains a proton at acidic pH, thus carrying a positive charge. This allows it to bind to negatively charged nucleic acid molecules or small molecule drugs via electrostatic interactions, and then self-assemble with auxiliary lipids to form lipid nanoparticles for gene drug delivery. The hydrophobic tail has 2-4 more tail groups than commonly used dual-tailed lipids in existing technologies. Due to the increased cross-section of the tail region, this lipid produces a more conical structure, giving it stronger endosome disruption capabilities and enhancing delivery efficiency. Furthermore, based on the amino acid-synthesized tail, it exhibits excellent biocompatibility. Addressing the low efficiency and high toxicity issues encountered in current gene drug delivery methods, this ionizable lipid with an amino acid backbone balances degradability and lipid safety while maintaining overall delivery efficiency in its chemical structure design. In addition, the preparation method of the amino acid backbone ionized lipids of the present invention is different from the harsh and complex synthetic route of traditional cationic lipids. The ionizable lipid library can be obtained through Michael addition, which has the advantages of simple synthetic route, clear reaction mechanism and easy high-throughput screening.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides an amino acid backbone ionizable lipid. Formula (A); Formula (B);

[0008] Wherein, R1 is selected from , C2-C10 alkyl, C2-C10 heteroalkyl, aralkyl, or absent;

[0009] R2 is selected from C3-C24 straight-chain alkyl, C4-C24 straight-chain alkenyl, ... ; n1=2~3, n2=3~24, n3=3~24;

[0010] X is O or NH;

[0011] R3 is selected from , , , , , , , , , , , C1-C10 hydroxyalkyl; R6 and R7 are each independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl;

[0012] R4 is selected from C1-C4 hydroxyalkyl; R5 is C1-C4 alkyl, C1-C4 hydroxyalkyl, n4 = 2~3

[0013] The asterisk (*) indicates a connection point.

[0014] The term "isomer" refers to the fact that the compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention envisions all such compounds, including cis and trans isomers, ( ) and (+) Enantiomers, (R) and (S) Enantiomers, diastereomers, (D) Isomer, (L) Isomers, racemic mixtures thereof, and other mixtures, such as enantiomers or diastereomer-enriched mixtures, are all within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0015] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other. Unless otherwise stated, the terms "cis-trans isomer" or "geometric isomer" arise from the inability of double bonds or single bonds on cyclic carbon atoms to rotate freely. Unless otherwise stated, the term "diastereomer" refers to stereoisomers of molecules having two or more chiral centers and being non-mirror images of each other. Unless otherwise stated, "(D)" or "(+)" indicates dextrorotatory, and "(L)" or "(...]" indicates... "(DL)" indicates left-handed rotation, and "(±)" or "(±)" indicates racemic rotation. Unless otherwise specified, use wedge-shaped solid lines. and wedge-shaped dashed key The absolute configuration representing the center of a solid.

[0016] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0017] Preferably, the structural formula of the amino acid backbone ionizable lipid is selected from any of the structures shown in formula (1) to formula (104):

[0018] Equation (1) Equation (2);

[0019] Equation (3) Equation (4);

[0020] Equation (5) Equation (6);

[0021] Equation (7) Equation (8);

[0022] Equation (9) Equation (10);

[0023] Equation (11) Equation (12);

[0024] Equation (13) Equation (14);

[0025] Equation (15) Equation (16);

[0026] Equation (17) Equation (18);

[0027] Equation (19) Equation (20);

[0028] Equation (21) Equation (22);

[0029] Equation (23) Equation (24);

[0030] Equation (25) Equation (26);

[0031] Equation (27); Equation (28);

[0032] Equation (29) Equation (30);

[0033] Equation (31) Equation (32);

[0034] Equation (33) Equation (34);

[0035] Equation (35) Equation (36);

[0036] Equation (37) Equation (38);

[0037] Equation (39) Equation (40);

[0038] Equation (41) Equation (42);

[0039] Equation (43) Equation (44);

[0040] Equation (45) Equation (46);

[0041] Equation (47) Equation (48);

[0042] Equation (49) Equation (50);

[0043] Equation (51) Equation (52);

[0044] Equation (53) Equation (54);

[0045] Equation (55) Equation (56);

[0046] Equation (57) Equation (58);

[0047] Equation (59) Equation (60);

[0048] Equation (61) Equation (62);

[0049] Equation (63); Equation (64);

[0050] Equation (65) Equation (66);

[0051] Equation (67) Equation (68);

[0052] Equation (69) Equation (70);

[0053] Equation (71) Equation (72);

[0054] Equation (73) Equation (74);

[0055] Equation (75) Equation (76);

[0056] Equation (77) Equation (78);

[0057] Equation (79) Equation (80);

[0058] Equation (81) Equation (82);

[0059] Equation (83) Equation (84);

[0060] Equation (85) Equation (86);

[0061] Equation (87) Equation (88);

[0062] Equation (89) Equation (90);

[0063] Equation (91) Equation (92);

[0064] Equation (93) Equation (94);

[0065] Equation (95) Equation (96);

[0066] Equation (97) Equation (98);

[0067] Equation (99) Equation (100);

[0068] Equation (101) Equation (102);

[0069] Equation (103) Equation (104).

[0070] The second aspect of the present invention provides a method for preparing the amino acid backbone ionizable lipid described in the first aspect, comprising the following steps: reacting a tail compound containing an amino acid structure or its stereoisomers or tautomers with an organic amine compound via a Michael addition reaction to obtain the amino acid backbone ionizable lipid;

[0071] The structural formula of the tail compound containing the amino acid structure is shown in formula (a): Equation (a); R1, R2, and X are as described in the first aspect.

[0072] Preferably, the Michael addition reaction is carried out at a temperature of 70-110°C.

[0073] More preferably, the reaction temperature of the Michael addition reaction is 80~100℃.

[0074] More preferably, the reaction temperature of the Michael addition reaction is 85~95℃.

[0075] Preferably, the Michael addition reaction takes place over a period of 36 to 60 hours.

[0076] More preferably, the Michael addition reaction takes place over a period of 40 to 56 hours.

[0077] More preferably, the Michael addition reaction takes place over a period of 44 to 52 hours.

[0078] Preferably, the organic amine compound is selected from one of the following compounds:

[0079]

[0080]

[0081] .

[0082] Preferably, the method for preparing the tail compound containing the amino acid skeleton includes the following steps: reacting chlorinated acrylate with compound a1 Tail compounds containing an amino acid backbone were prepared by esterification.

[0083] More preferably, the reaction temperature of the esterification reaction is 15~40°C.

[0084] More preferably, the reaction temperature of the esterification reaction is 20~35℃.

[0085] More preferably, the esterification reaction takes 12 to 24 hours.

[0086] More preferably, the esterification reaction is carried out at a reaction temperature of 12-16 h.

[0087] More preferably, the reaction system for the esterification reaction further includes an organic base catalyst.

[0088] More preferably, the organic base catalyst is triethylamine.

[0089] More preferably, the molar ratio of chlorinated acrylate to compound a1 is (1~2):1.

[0090] More preferably, the molar ratio of chlorinated acrylate to compound a1 is (1~1.5):1.

[0091] More preferably, the preparation method of compound a1 includes the following steps: reacting a Boc-amino acid with an alkyl alcohol or an enyl alcohol, and then reacting the reaction product with trifluoroacetic acid to undergo a deprotection reaction to obtain compound a1.

[0092] More preferably, the reaction system further includes an activator and an acid promoter.

[0093] More preferably, the active agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI).

[0094] More preferably, the acid accelerator comprises 4-dimethylaminopyridine (DMAP).

[0095] More preferably, the reaction temperature is 15~40℃.

[0096] More preferably, the reaction temperature of the deprotection reaction is 15~40℃.

[0097] A third aspect of the present invention provides a lipid composition comprising the amino acid backbone ionizable lipid, sterol, and polyethylene glycol-modified lipid described in the first aspect.

[0098] Preferably, it also includes neutral auxiliary lipids.

[0099] More preferably, the neutral auxiliary lipid includes at least one of distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilinoleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and palmitoylphosphatidylcholine (POPC).

[0100] Preferably, the polyethylene glycol (PEG)-modified lipids include at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0101] Preferably, the sterol includes at least one of cholesterol and β-sitosterol.

[0102] More preferably, when the lipid composition further includes cholesterol, cofactor phospholipids and polyethylene glycol-modified lipids, the molar ratio of the amino acid backbone to ionizable lipids: cholesterol: cofactor phospholipids: polyethylene glycol-modified lipids is (30-50): (35-60): (5-20): (1-5).

[0103] The fourth aspect of the present invention provides the use of the amino acid backbone ionizable lipids described in the first aspect, or the lipid composition described in the third aspect, in the preparation of a bioactive substance delivery system, wherein the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle, or microbubble.

[0104] A fifth aspect of the present invention provides a pharmaceutical composition comprising the lipid composition described in the third aspect and an active substance; said active substance comprising at least one of nucleic acid molecules, small molecule compounds, polypeptides, and proteins.

[0105] Preferably, the mixture includes a carrier and an active substance, wherein the carrier comprises the lipid composition described in the third aspect; the active substance is encapsulated within the carrier or bound to the carrier.

[0106] More preferably, the method for preparing the pharmaceutical composition includes the following steps:

[0107] The amino acid backbone ionizable lipids, sterols, neutral auxiliary lipids, and polyethylene glycol-modified lipids are mixed in an ethanol solvent to prepare a lipid mixture solution; the active substance is mixed with an acidic buffer solution, and then mixed with the lipid mixture solution and incubated to obtain a drug carrier, i.e., the drug composition.

[0108] Alternatively, the ionizable lipids with the amino acid backbone, sterols, and neutral auxiliary lipids are dissolved in chloroform, dried with nitrogen to evaporate the solvent, and mixed with acidic or neutral buffer solution to prepare liposome nanoparticles for later use; cationic polypeptides and active substances are mixed, then mixed with the liposome nanoparticles, and then polyethylene glycol-modified lipids are added to prepare a drug carrier.

[0109] More preferably, the cationic polypeptide is protamine.

[0110] More preferably, the acidic buffer solution has a pH of 3-7; the acidic buffer solution is a sodium acetate or sodium citrate buffer solution.

[0111] Preferably, when the active pharmaceutical ingredient includes nucleic acid molecules, the nitrogen-to-phosphorus ratio of the ionizable lipids in the amino acid backbone to the nucleic acid molecules is (1~100):1.

[0112] More preferably, when the active pharmaceutical ingredient includes nucleic acid molecules, the nitrogen-to-phosphorus ratio of the ionizable lipids in the amino acid backbone to the nucleic acid molecules is (1~30):1.

[0113] More preferably, when the active pharmaceutical ingredient includes nucleic acid molecules, the nitrogen-to-phosphorus ratio of the ionizable lipids in the amino acid backbone to the nucleic acid molecules is (1~10):1.

[0114] Preferably, the nucleic acid molecule includes at least one of siRNA, miRNA, mRNA, circRNA, antisense RNA, CRISPR guide RNAs, replicable RNA, circular dinucleotide, polyIC, CpG ODN, plasmid DNA, and microcircular DNA.

[0115] Preferably, the protein includes at least one of cell colony-stimulating factor, interleukin, lymphotoxin, interferon-like protein, tumor necrosis factor, antibody, and protein antigen.

[0116] The sixth aspect of this invention provides the use of the amino acid backbone ionizable lipids described in the first aspect, or the pharmaceutical composition described in the fifth aspect, in the preparation of nucleic acid drugs, gene vaccines, polypeptide or protein drugs, and small molecule drugs.

[0117] The beneficial effects of this invention are:

[0118] (1) This invention proposes an ionizable lipid with an amino acid backbone, which is modified with amino acids as the core and has more ester groups and peptide bonds. After effectively releasing RNA in vivo, it can be rapidly hydrolyzed by enzymes and is easily metabolized and cleared in vivo, and has biodegradability. It has a four-tail structure, which can increase the cross-sectional area of ​​the lipid tail, help RNA and other drugs escape from the endosome, and thus enhance the transfection effect. The charge of the ionizable lipid can change with the pH of the environment. Under physiological conditions, it is electrically neutral, which reduces the cytotoxicity caused by excessive positive charge, thereby increasing the stability of lipid nanoparticles and helping to prolong the circulation time of the loaded nucleic acid drugs and improve the pharmacokinetic characteristics.

[0119] (2) The LNP made of ionizable lipids, auxiliary phospholipids, cholesterol and PEG lipids provided by the present invention has better nucleic acid carrier performance and can effectively deliver nucleic acid drugs such as siRNA, mRNA, pDNA into cells to exert their effects.

[0120] (3) The preparation method of the amino acid skeleton ionized lipid of the present invention is different from the harsh and complex synthetic route of traditional cationic lipids. The ionizable lipid library can be obtained through Michael addition, which has the advantages of simple synthetic route, clear reaction mechanism and easy high-throughput screening. Attached Figure Description

[0121] Figure 1 The relative luciferase activity results of cell transfection with different lipid nanoparticles are shown in the figure.

[0122] Figure 2 (a) Relative luciferase activity results of cell transfection with lipid nanoparticles of different neutral phospholipids; (b) Relative luciferase activity results of cell transfection with lipid nanoparticles of different component ratios.

[0123] Figure 3 (c) Relative luciferase activity results for cell transfection with lipid nanoparticles of different nitrogen-to-phosphorus ratios; (d) Relative luciferase activity results for cell transfection with lipid nanoparticles of different buffers.

[0124] Figure 4 Fluorescence microscopy for transfection of different lipid nanoparticles into Jurkat T cell lines;

[0125] Figure 5 Imaging images of different lipid nanoparticles transfected in mice;

[0126] Figure 6 The relative luciferase activity results of different lipid nanoparticles transfected in mice are shown in the figure.

[0127] Figure 7 The relative luciferase activity results of transfecting different lipid nanoparticles into the spleen and liver of mice in vivo are shown in the figure.

[0128] Figure 8 The graph shows the relative luciferase activity ratios in the spleen and liver of mice after transfection with different lipid nanoparticles. Detailed Implementation

[0129] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0130] The specific steps of the general method for preparing partially amino acid backbone ionizable lipids of the present invention are as follows:

[0131] (1) Synthesizing the hydrophobic tail of the amino acid backbone

[0132] The specific steps are as follows: In a 250 mL reaction flask, add 10 mmol of Boc-amino acid, 150 mL of dichloromethane (DCM), a magnetic flux, 22 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol of 4-dimethylaminopyridine (DMAP). Stir at room temperature for 15 minutes, then add 20 mmol of alkyl alcohol and react for 24 h until the reaction is complete. Transfer the reaction mixture to a separatory funnel, add DCM (2 x 100 mL), wash with 1 M HCl (2 x 100 mL), and extract with saturated brine (2 x 200 mL). Collect the organic layer, dry it with anhydrous magnesium sulfate, filter, and then remove the organic solvent using a rotary evaporator under reduced pressure. Separate the product by thin-layer chromatography.

[0133] Removal of amino protecting groups: The product was dissolved in 30 mL DCM, and 20 mL trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h. The organic solvent was removed by rotary evaporation under reduced pressure, and the solution was redissolved in 150 mL DCM. The solution was washed with saturated sodium bicarbonate (2 x 100 mL) and extracted with saturated brine (2 x 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed by rotary evaporation under reduced pressure. The product obtained did not require further purification and could be used for the next reaction.

[0134] (2) Synthesizing linking groups

[0135] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the amino acid backbone alkyl tail of the product synthesized in the previous step, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, then diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was dried over anhydrous magnesium sulfate and filtered. The product was separated by rapid chromatography.

[0136] (3) Reaction of head group and tail group

[0137] Select the alkyl tail synthesized in step (2) and 100 mg of amine, and add them sequentially to a 3 mL reaction flask lined with tetrafluoroethylene. The reaction is heated at 90 °C for 48 h. After the reaction is completed, the product can be directly used for cell transfection experiments or separated by rapid chromatography column separation.

[0138] The Boc-amino acids synthesized in this invention are L-type or D-type amino acids, thereby obtaining ionizable lipids with an amino acid skeleton containing L-type or D-type amino acid groups.

[0139] Example 1

[0140] Example 1 provides an ionizable lipid with an amino acid backbone, the specific preparation method of which is as follows:

[0141] (1) Synthesizing the hydrophobic tail of the amino acid backbone

[0142]

[0143] In a 250 mL reaction flask, 10 mmol of BOC-L-glutamic acid, 150 mL of dichloromethane (DCM), a magnetic flux, 22 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 20 mmol of oleyl alcohol. The reaction was allowed to proceed for 24 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) was added. The mixture was washed with 1 M HCl (2 x 100 mL) and extracted with saturated brine (2 x 200 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the target product A in 73% yield.

[0144]

[0145] Removal of amino protecting groups: Intermediate A was dissolved in 30 mL DCM, and 20 mL trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h. The organic solvent was removed by rotary evaporation under reduced pressure, and the solution was redissolved in 150 mL DCM. The solution was washed with saturated sodium bicarbonate (2 x 100 mL) and extracted with saturated brine (2 x 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed by rotary evaporation under reduced pressure to obtain product B. No further purification was required for the next reaction.

[0146] (2) Synthesizing linking groups

[0147]

[0148] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the intermediate product B amino acid skeleton alkyl tail, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and the reaction progress was monitored by TLC. Once the reaction was complete, the mixture was diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 5:1) to obtain the target product C in 95% yield.

[0149] The proton NMR data of the obtained product are as follows:

[0150] 1 H NMR (400 MHz, CDCl3): 6.37 (d, J = 3.6, 1H), 6.30 (d, J = 16 Hz,1H), 5.79 (m, 1H), 5.6(d, J = 10.4 Hz, 1H), 5.38-5.29 (m, 4H), 4.72-4.67 (m,1H), 4.15-4.02 (dt, J = 6.8 Hz, 4H), 2.43-2.32 (m, 2H), 2.27-2.19 (m, 1H), 2.08-1.95 (m, 7H), 1.29-1.25 (m, 42H), 1.55-1.36 (m, 8H), 0.87 (t, J = 13.6Hz, 6H).

[0151] (3) Reaction of head group and tail group

[0152]

[0153] 100 mg of 3-aminopropanol and 2 stoichiometric amounts of intermediate C were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner), and the reaction was carried out at 90 °C for 48 h. After the reaction was completed, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 29GluOC18U.

[0154] The proton NMR data of the obtained product are as follows:

[0155] 1H NMR (400 MHz, CDCl3): 4.78-4.65 (m, 4H), 4.08-4.03 (m, 4H), 3.19-3.02 (m, 8H), 2.81-2.77 (m, 2H), 2.63-2.42 (m, 10H), 1.80-1.48 (m, 20H), 1.39-1.25 (m, 20H), 0.87 (dd, J1= 6.8 Hz, J2= 5.2 Hz, 12H).

[0156] Example 2

[0157] Example 2 provides an ionizable lipid with an amino acid backbone, the preparation method of which is as follows:

[0158]

[0159] 100 mg of 5-aminopentanol and 2 stoichiometric amounts of intermediate C were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner), and the reaction was carried out at 90 °C for 48 h. After the reaction was completed, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 34GluOC18U.

[0160] The proton NMR data of the obtained product are as follows:

[0161] 1 H NMR (400 MHz, CDCl3): 7.73-7.38 (m, 2H), 5.41-5.35 (m, 8H), 4.58-4.53 (m, 2H), 4.14-4.06 (m, 8H), 3.69-3.66 (m, 2H), 3.16-3.10 (m, 8H), 2.81-2.77 (m, 2H), 2.47-2.41 (m, 4H), 2.24-1.98 (m, 20H), 1.67-1.59 (m, 10H), 1.45-1.41 (m, 4H), 1.36-1.27 (m, 88H), 0.91-0.88 (m, 12H).

[0162] Example 3

[0163] Example 3 provides an ionizable lipid with an amino acid backbone, the preparation method of which is as follows:

[0164] (1) Synthesizing linking groups

[0165]

[0166] In a three-necked flask equipped with a magnetic stir bar, 10 mmol L-aspartic acid, 30 mmol TEA, and 120 mL DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 11 mmol acryloyl chloride (premixed in 20 mL dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and the reaction progress was monitored by TLC. Once the reaction was complete, the product was diluted with 50 mL DCM and washed with 1 M HCl (2 × 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. Product D was used directly without purification.

[0167] (2) Synthesizing the hydrophobic tail of the amino acid backbone

[0168]

[0169] In a 100 mL reaction flask, 5 mmol of intermediate D, 50 mL of dichloromethane (DCM), a magnetic flux, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 5 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 11 mmol of n-octanol. The reaction was allowed to proceed for 24 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) was added. The mixture was washed with 1 M HCl (2 x 100 mL) and extracted with saturated brine (2 x 200 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 20:1) to give the target product E in 43% yield.

[0170] The proton NMR data of the obtained product are as follows:

[0171] 1 H NMR (400 MHz, CDCl3): 6.69 (d, J = 8 Hz, 1H), 6.32 (d, J = 8 Hz,1H), 6.2-6.13 (m, 1H), 5.7(d, J = 10.4 Hz, 1H), 4.93-4.89 (m, 1H), 4.19-4.06(m, 4H), 3.10-2.88 (m, 2H), 1.65-1.59 (m, 4H), 1.34-1.21 (m, 20H), 0.89 (t, J= 6 Hz, 6H).

[0172] (2) Reaction of head group and tail group

[0173]

[0174] 100 mg of N,N-dimethylethylenediamine and 2 stoichiometric amounts of intermediate E were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner). The reaction was carried out at 90 °C for 48 h. After the reaction was complete, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 1AspOC8.

[0175] The proton NMR data of the obtained product are as follows:

[0176] 1 H NMR (400 MHz, CDCl3): 4.78-4.65 (m, 4H), 4.08-4.03 (m, 4H), 3.19-3.02 (m, 8H), 2.81-2.77 (m, 2H), 2.63-2.42 (m, 10H), 1.80-1.48 (m, 20H), 1.39-1.25 (m, 20H), 0.87 (dd, J1= 6.8 Hz, J2= 5.2 Hz, 12H).

[0177] Example 4

[0178] Example 4 provides an ionizable lipid with an amino acid backbone, the specific preparation method of which is as follows:

[0179] (1) Synthesizing the hydrophobic tail of the amino acid backbone

[0180]

[0181] In a 250 mL reaction flask, 10 mmol of BOC-L-aspartic acid, 150 mL of dichloromethane (DCM), a magnetic flux, 22 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 20 mmol of 9-nonadecanol. The reaction was allowed to proceed for 24 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) was added. The mixture was washed with 1 M HCl (2 x 100 mL) and extracted with saturated brine (2 x 200 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 30:1) to obtain the target product F in 68% yield.

[0182]

[0183] Removal of amino protecting groups: Intermediate F was dissolved in 30 mL DCM, and 20 mL trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h. The organic solvent was removed by rotary evaporation under reduced pressure, and the solution was redissolved in 150 mL DCM. The solution was washed with saturated sodium bicarbonate (2 x 100 mL) and extracted with saturated brine (2 x 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed by rotary evaporation under reduced pressure to obtain product G. No further purification was required for the next reaction.

[0184] (2) Synthesizing linking groups

[0185]

[0186] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the intermediate G amino acid skeleton alkyl tail, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and the reaction progress was monitored by TLC. Once the reaction was complete, the mixture was diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 8:1) to obtain the target product H in 88% yield.

[0187] The proton NMR data of the obtained product are as follows:

[0188] 1 H NMR (400 MHz, CDCl3): 6.69 (m, 1H), 6.32 (d, J = 16.8 Hz, 1H), 6.19-6.12 (m, 1H), 5.68 (d, J = 10 Hz, 1H), 4.93-4.89 (m, 1H), 4.08-3.93 (m,4H), 3.10-2.89 (m, 2H), 1.63-1.59 (m, 2H), 1.43-1.26 (m, 66H), 0.88 (t, J =6.4 Hz, 12H).

[0189] (3) Reaction of head group and tail group

[0190]

[0191] 100 mg of 1-(2-aminoethyl)piperidine and two stoichiometric amounts of intermediate product H were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner). The reaction was carried out at 90 °C for 48 h. After the reaction was completed, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 13AspOC8C10.

[0192] The proton NMR data of the obtained product are as follows:

[0193] 1 H NMR (400 MHz, CDCl3): 4.78-4.65 (m, 4H), 4.08-4.03 (m, 4H), 3.19-3.02 (m, 8H), 2.81-2.77 (m, 2H), 2.63-2.42 (m, 10H), 1.80-1.48 (m, 20H), 1.39-1.25 (m, 20H), 0.87 (dd, J1= 6.8 Hz, J2= 5.2 Hz, 12H).

[0194] Example 5

[0195] Example 5 provides an ionizable lipid with an amino acid backbone, the specific preparation method of which is as follows:

[0196] (1) Synthesizing the hydrophobic tail of the amino acid backbone

[0197]

[0198] In a 250 mL reaction flask, 10 mmol of BOC-L-aspartic acid, 150 mL of dichloromethane (DCM), a magnetic flux, 22 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 20 mmol of oleylamine. The reaction was allowed to proceed for 24 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) was added. The mixture was washed with 1 M HCl (2 x 100 mL) and extracted with saturated brine (2 x 200 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 4:1) to obtain the target product I in 73% yield.

[0199]

[0200] Removal of amino protecting groups: Intermediate I was dissolved in 30 mL DCM, and 20 mL trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h. The organic solvent was removed by rotary evaporation under reduced pressure, and the solution was redissolved in 150 mL DCM. The solution was washed with saturated sodium bicarbonate (2 x 100 mL) and extracted with saturated brine (2 x 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed by rotary evaporation under reduced pressure to obtain product J. No further purification was required for the next reaction.

[0201] (2) Synthesizing linking groups

[0202]

[0203] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the intermediate product J (amino acid skeleton alkyl tail), 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and the reaction progress was monitored by TLC. Once the reaction was complete, the mixture was diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 2:1) to obtain the target product K in 86% yield.

[0204] The proton NMR data of the obtained product are as follows:

[0205] 1 H NMR (400 MHz, CDCl3): 7.88 (d, J = 7.2 Hz, 1H), 7.49 (t, J = 5.6Hz, 1H), 6.76 (t, J = 5.2 Hz, 1H), 6.33-6.24 (m, 1H), 5.67 (d, J = 9.6 Hz,1H), 5.40-5.33 (m, 4H), 4.82-4.79 (m, 1H), 3.23-3.17 (m, 4H), 2.86-2.81 (m,1H), 2.57-2.52 (m, 1H), 2.03-2.0 (m, 8H), 1.37-1.18 (m, 48H), 0.88 (dd, J =6.4 Hz, 6H).

[0206] (3) Reaction of head group and tail group

[0207]

[0208] 100 mg of 2-(2-methyl-1H-imidazol-1-yl)ethylamine and 2 stoichiometric amounts of intermediate K were added to a 5 mL reaction flask equipped with a magnetic inlet (cap lined with tetrafluoroethylene). The reaction was carried out at 90 °C for 48 h. After the reaction was complete, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 1AspNC18U.

[0209] The proton NMR data of the obtained product are as follows:

[0210] 1 H NMR (400 MHz, CDCl3): 4.78-4.65 (m, 4H), 4.08-4.03 (m, 4H), 3.19-3.02 (m, 8H), 2.81-2.77 (m, 2H), 2.63-2.42 (m, 10H), 1.80-1.48 (m, 20H), 1.39-1.25 (m, 20H), 0.87 (dd, J1= 6.8 Hz, J2= 5.2 Hz, 12H).

[0211] Example 6

[0212] Example 6 provides an ionizable lipid with an amino acid backbone, the specific preparation method of which is as follows:

[0213] (1) Synthesizing the hydrophobic tail of the amino acid backbone

[0214]

[0215] In a 250 mL reaction flask, 10 mmol of BOC-L-valine, 150 mL of dichloromethane (DCM), a magnetic flux, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 10 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 10 mmol of oleyl alcohol. The reaction was allowed to proceed for 24 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) was added. The mixture was washed with 1 M HCl (2 x 100 mL) and extracted with saturated brine (2 x 200 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the target product L in 77% yield.

[0216]

[0217] Removal of amino protecting groups: Intermediate L was dissolved in 30 mL DCM, and 15 mL trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h. The organic solvent was removed by rotary evaporation under reduced pressure, and the solution was redissolved in 150 mL DCM. The solution was washed with saturated sodium bicarbonate (2 x 100 mL) and extracted with saturated brine (2 x 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed by rotary evaporation under reduced pressure to obtain product M. No further purification was required for the next reaction.

[0218] (2) Synthesizing linking groups

[0219]

[0220] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the intermediate product M (amino acid backbone alkyl tail), 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and the reaction progress was monitored by TLC. Once the reaction was complete, the mixture was diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the target product N in 98% yield.

[0221] The proton NMR data of the obtained product are as follows:

[0222] 1 H NMR (400 MHz, CDCl3): 6.41-6.30 (m, 1H), 6.22-6.15 (m, 1H), 5.68-5.66 (m, 1H), 5.41-5.30 (m, 2H), 4.69-4.66 (m, 1H), 4.17-4.10 (m, 1H), 2.23-1.95 (m, 5H), 1.68-1.61 (m, 2H), 1.33-1.26 (m, 22H), 0.97-0.85 (m, 9H).

[0223] (3) Reaction of head group and tail group

[0224]

[0225] 100 mg of 3-aminopropanol and 2 stoichiometric amounts of intermediate product N were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner), and the reaction was carried out at 90 °C for 48 h. After the reaction was completed, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 17ValOC18U.

[0226] The proton NMR data of the obtained product are as follows:

[0227] 1 H NMR (400 MHz, CDCl3): 8.05-7.83 (m, 1H), 7.39-7.37 (m, 1H), 7.21-7.14 (m, 1H), 7.09-7.01 (m, 1H), 5.40-5.34 (m, 4H), 4.58-4.50 (m, 2H), 4.16-4.01 (m, 6H), 3.15-3.10 (q, J= 8.0 Hz, 4H), 2.96-2.67 (m, 4H), 2.53-2.40 (m,4H), 2.23-1.95 (m, 10H), 1.66-1.61 (m, 4H), 1.42-1.26 (m, 44H), 0.97-0.87 (m, 18H)).

[0228] Example 7

[0229] Example 7 provides an ionizable lipid with an amino acid backbone, the preparation method of which is as follows:

[0230] (1) Synthesizing the hydrophobic tail of the amino acid backbone

[0231]

[0232] In a 250 mL reaction flask, 10 mmol of BOC-L-methionine, 150 mL of dichloromethane (DCM), a magnetic flux, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 10 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 10 mmol of oleyl alcohol. The reaction was allowed to proceed for 24 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) was added. The mixture was washed with 1 M HCl (2 x 100 mL) and extracted with saturated brine (2 x 200 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the target product O in 64% yield.

[0233]

[0234] Removal of amino protecting groups: Intermediate O was dissolved in 30 mL DCM, and 15 mL trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h. The organic solvent was removed by rotary evaporation under reduced pressure, and the solution was redissolved in 150 mL DCM. The solution was washed with saturated sodium bicarbonate (2 x 100 mL) and extracted with saturated brine (2 x 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed by rotary evaporation under reduced pressure to obtain product P. No further purification was required for the next reaction.

[0235] (2) Synthesizing linking groups

[0236]

[0237] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the intermediate product M (amino acid skeleton alkyl tail), 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and the reaction progress was monitored by TLC. Once the reaction was complete, the mixture was diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the target product Q in 88% yield.

[0238] The proton NMR data of the obtained product are as follows:

[0239] 1 H NMR (400 MHz, CDCl3): 6.34-6.30 (m, 1H), 6.20-6.13 (m, 1H), 5.70-5.67 (m, 1H), 5.40-5.30 (m, 2H), 4.82-4.77 (m, 2H), 4.16-4.11 (m, 1H), 2.59-2.47 (m, 2H), 2.26-1.94 (m, 9H), 1.68-1.61 (m, 2H), 1.34-1.23 (m, 22H), 0.89-0.85 (m, 3H).

[0240] (3) Reaction of head group and tail group

[0241]

[0242] 100 mg of 3-aminopropanol and 2 stoichiometric amounts of intermediate product Q were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner), and the reaction was carried out at 90 °C for 48 h. After the reaction was completed, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 17ValOC18U.

[0243] The proton NMR data of the obtained product are as follows:

[0244] 1H NMR (400 MHz, CDCl3): 7.77-7.69 (m, 2H), 5.41-5.35 (m, 4H), 4.69-4.62 (m, 2H), 4.15-4.11 (m, 4H), 3.80-3.76 (m, 2H), 3.18-3.10 (m, 4H), 2.84-2.71 (m, 4H), 2.62-2.57 (m, 4H), 2.14-1.97 (m, 20H), 1.68-1.64 (m, 4H), 1.45-1.27 (m, 44H), 0.91-0.88 (m, 6H).

[0245] Example 8

[0246] Example 8 provides an ionizable lipid with an amino acid backbone, the preparation method of which is as follows:

[0247] (1) Synthesizing the hydrophobic tail of the amino acid backbone

[0248]

[0249] In a 250 mL reaction flask, 10 mmol of BOC-L-phenylalanine, 150 mL of dichloromethane (DCM), a magnetic flux, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 10 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 10 mmol of oleyl alcohol. The reaction was allowed to proceed for 24 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) was added. The mixture was washed with 1 M HCl (2 x 100 mL) and extracted with saturated brine (2 x 200 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the target product R in 97% yield.

[0250]

[0251] Removal of amino protecting groups: Intermediate R was dissolved in 30 mL DCM, and 15 mL trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h. The organic solvent was removed by rotary evaporation under reduced pressure, and the solution was redissolved in 150 mL DCM. The solution was washed with saturated sodium bicarbonate (2 x 100 mL) and extracted with saturated brine (2 x 100 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed by rotary evaporation under reduced pressure to obtain product S. No further purification was required for the next reaction.

[0252] (2) Synthesizing linking groups

[0253]

[0254] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the intermediate product S (amino acid skeleton alkyl tail), 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and the reaction progress was monitored by TLC. Once the reaction was complete, the mixture was diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the target product T in 69% yield.

[0255] The proton NMR data of the obtained product are as follows:

[0256] 1 H NMR (400 MHz, CDCl3): 7.31-7.11 (m, 5H), 6.33-6.27 (m, 1H), 6.16-6.09 (m, 1H), 5.68-5.65 (m, 1H), 5.41-5.35 (m, 2H), 4.99-4.95 (m, 1H), 4.15-4.08 (m, 2H), 3.19-3.16 (m, 2H), 2.06-2.03 (m, 4H), 1.63-1.59 (m, 2H), 1.37-1.28 (m, 22H), 0.91-0.87 (m, 3H).

[0257] (3) Reaction of head group and tail group

[0258]

[0259] 100 mg of 3-aminopropanol and 2 stoichiometric amounts of intermediate product T were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner), and the reaction was carried out at 90 °C for 48 h. After the reaction was completed, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 34PheOC18U.

[0260] The proton NMR data of the obtained product are as follows:

[0261] 1 H NMR (400 MHz, CDCl3): 7.31-7.16 (m, 10H), 5.41-5.35 (m, 4H), 4.84-4.80 (m, 2H), 4.10-4.06 (m, 4H), 3.66-3.63 (m, 4H), 3.66-3.05 (m, 6H), 2.95-2.94 (m, 2H), 2.65-2.52 (m, 4H), 2.08-1.96 (m, 8H), 1.62-1.53 ​​(m, 6H), 1.45-1.27 (m, 48H), 0.91-0.88 (m, 6H).

[0262] The ionizable lipids with amino acid backbones synthesized in this invention are as follows (the synthesis methods for ionizable lipids with other amino acid backbone structures are described in the general preparation methods and Examples 1-8 above): 1GluOC8, 1AspOC8, 1GluOC10, 1AspOC10, 1GluOC8C10, 1AspOC8C10, 1GluOC10, 1AspOC10, 1GluOC12, 1AspOC12, 1GluOC16, 1AspOC16, 1GluOC18, 1AspOC18, 1GluOC18U, 1AspOC18U, 1GluOC18U2, 1AspOC18U2, 1GluNC8, 1AspNC8, 1G luNC18U, 1AspNC18U, 3GluOC18U, 3AspOC18U, 4GluOC18U, 4AspOC18U, 1LeuOC18U, 1LeuNC18U, 6AspOC18U, 6GluOC18U, 8GluOC18, 8AspOC18, 8GluOC18 U, 9GluOC8, 8AspOC18U, 9AspOC8, 9GluOC10, 9AspOC10, 9GluOC18U, 9AspOC18U, 9GluOC18U2, 9AspOC18U2, 11GluOC8, 11AspOC8, 11GluOC10, 11AspOC10 , 12GluOC18U, 12AspOC18U, 13AspOC8, 13AspOC10, 13GluOC8C10, 13AspOC8C10, 16AspOC10, 16GluOC18U2, 16GluOC18U, 16AspOC18U, 17AspOC10, 17Gl uOC18U2, 17GluOC18U, 17AspOC18U, 18GluOC18U, 18AspOC18U, 19GluOC8C10, 19AspOC8C10, 19GluOC18U, 19AspOC18U, 20GluOC18U, 20AspOC18U, 21Gl uOC18U, 21GluNC18U, 22GluOC18U, 22AspOC18U, 24GluOC18U, 24AspOC18U, 25GluOC18U, 25AspOC18U, 26GluOC18U, 26AspOC18U, 27GluOC18U, 27AspOC 18U, 28GluOC18U, 28AspOC18U, 29GluOC18U, 29AspOC18U, 30GluOC18U, 30AspOC18U, 31GluOC18U, 31AspOC18U, 32GluOC18U, 32GluNC18U, 33GluOC18U,33AspOC18U, 34GluOC18U, 34AspOC18U, 17PheOC18, 17MetOC18U, 34ValOC18U, 17GayOC18U, 29PheO C18U, 29MetOC18U, 29ValOC18U, 29GayOC18U, 34PheOC18U, 34MetOC18U, 34ValOC18U, 34GayOC18U. ,

[0263] lipid experimental characterization

[0264] 1. Characterization of enzyme delivery efficiency of fluorescent proteins and luciferins

[0265] The efficiency of delivering a plasmid (DNA-GFP-Luc) encoding green fluorescent protein (GFP) and firefly luciferase (Luc) using LNPs containing ionizable lipids was verified in the 293T cell line. The ionizable lipids used were 1GluOC18, 8GluOC18, 3GluOC18U, 4GluOC18U, 12GluOC18U, 13GluOC18U, 17GluOC18U, 1AspOC18U, 3AspOC18U, 1AspOC8, 1AspOC10, 8GluOC18U, 9AspOC8, 9AspOC10, 9GluOC18U, 13AspOC8, and 13AspOC10. , 13AspOC8C10, 11AspOC8, 11AspOC10, 16AspOC10, 16AspOC18U, 17AspOC10, 17AspOC18U, 19AspOC18U , 19AspOC8C10, 22AspOC18U, 21G;uNC18U, 16GluOC18U, 20GluOC18U, 21GluOC18U, 1GluOC18U2, 9GluOC 18U2, 16GluOC18U2, 17GluOC18U2, 9GluOC18U, 16GluOC18U, 19GluOC18U, 25GluOC18U, 26GluOC18U, 2 7GluOC18U, 29GluOC18U, 30GluOC18U, 31GluOC18U, 32GluOC18U, 33GluOC18U, 34GluOC18U, 17GayOC18 U, 29GayOC18U, 34GayOC18U, 17PheOC18U, 29PheOC18U, 34PheOC18U, 17ValOC18U, 29ValOC18U, 34ValOC18U, 17MetOC18U, 29MetOC18U, 34MetOC18U, and the commercially available materials ALC-0315 and SM-102 were used as delivery materials to express DNA-GFP-Luc in cells.

[0266] Specific steps:

[0267] (1) Cell culture: The day before the experiment, the cultured 293T cells were seeded in a 96-well cell culture plate. When the cell density grew to about 70-80%, the cell transfection experiment was carried out.

[0268] (2) Preparation of lipid nanoparticles LNP-DNA-GFP-Luc for cell transfection

[0269] The aforementioned ionizable lipids were dissolved in anhydrous ethanol at concentrations of 10, 3, 6, and 1 mg / mL, respectively, along with DSPC, Cholesterol, and DSPE-PEG. The solutions were then uniformly mixed at a molar ratio of ionizable lipids:Cholesterol:DSPC:DSPE-PEG = 40:48:10:2. Simultaneously, an appropriate amount of DNA-GFP-Luc was dissolved in sodium citrate buffer (three times the total volume of the lipid mixture, pH 4.0–4.5). The DNA buffer and lipid mixture were then rapidly mixed and incubated at room temperature for 15 min to assemble stable LNPs (each well containing 150 ng of DNA-GFP-Luc). The LNPs were diluted with twice the volume of sterile PBS and added to 96-well cell culture plates for transfection. The nitrogen-to-phosphorus ratio of the ionizable lipids to nucleic acids was 6:1, which is the molar ratio between protonated amino groups and phosphate groups on the nucleic acids (the same applies below).

[0270] Positive control group: LNPs were assembled using commercially available lipids ALC-0315 and SM-102 according to published preparation methods. The specific procedures were as follows: ALC-0315 or SM-102, DSPC, Cholesterol, ALC-0159, or DMG-PEG were added. 2000 Dissolve the compounds in anhydrous ethanol at concentrations of 5 mg / mL, 1.5 mg / mL, 3 mg / mL, and 1 mg / mL, respectively, according to a ratio of ALC-0315:Cholesterol:DSPC:ALC-0159 = 46.3:42.7:9.4:1.6 or SM-102:Cholesterol:DSPC:DMG-PEG. 2000 The lipid mixture was thoroughly mixed in a molar ratio of 50:38.5:10:1.5. Simultaneously, an appropriate amount of DNA-GFP-Luc was dissolved in sodium citrate buffer (the volume of the sodium citrate buffer was three times the total volume of the lipid mixture, pH=4.0). The DNA buffer and lipid mixture solution were then rapidly mixed and incubated at room temperature for 15 min to assemble stable LNPs (each well contained 150 ng of DNA-GFP-Luc LNPs). The LNPs were diluted with twice the volume of sterile PBS and added to 96-well cell culture plates for transfection. The nitrogen-phosphorus ratio of ALC-0315, SM-102, and nucleic acids was 6:1.

[0271] Negative control group: 293T cells were cultured normally and then added with unloaded DNA-GFP-Luc.

[0272] (3) Cell transfection efficiency analysis

[0273] Thirty-six hours after cell transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. The culture medium in the 96-well cell culture plates was aspirated, cell lysis buffer was added, and cells were lysed on ice for 30 min. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are as follows: Figure 1 As shown in the figure. The results show that the ionizable lipids synthesized in this invention can greatly enhance the transfection efficiency of nucleic acids. When the tail of the ionizable lipid is GluO18U or AspOC18U, the RNA expression efficiency is the highest, while when the tail is GluO8 or AspOC8, the RNA expression efficiency is weaker. The transfection efficiency of lipids represented by 29GluO18U, 34GluO18U, and 17AspOC18U is better than that of commercial lipids ALC-0315 and SM-102, with an efficiency improvement of about 2 to 3 times, which verifies the rationality and high efficiency of the overall chemical structure of the ionizable lipids designed in this invention.

[0274] 2. LNP component optimization experiment

[0275] The efficiency of LNP containing ionizable lipids in delivering saRNA-GFP-Luc was validated in the 293T cell line. The composition of the LNP was optimized using the ionizable lipid 17LGluO18U.

[0276] Specific steps:

[0277] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was 17GluO18U. The ionizable lipids, DOPE, DSPC or DOPC, Cholesterol or β-sitosterol, DSPE-PEG or DMG-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL and 1 mg / mL, respectively. The ratio of ionizable lipid 17GluO18U: (Cholesterol or β-sitosterol): (DOPE, DSPC or DOPC): (DSPE-PEG or DMG-PEG) = 40:48:10:2.

[0278] (2) Cell transfection efficiency analysis

[0279] Thirty-six hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. The culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are as follows: Figure 2As shown in (a) above. The results show that the chemical structure of neutral helper phospholipids greatly affects RNA delivery efficiency. When the neutral helper phospholipid is DSPC, the delivery efficiency of all three ionizable lipids is significantly better than that of DOPE or DOPC. Therefore, DSPC is the preferred neutral helper phospholipid. Cholesterol is preferred over β-sitosterol. The chain length of PEG significantly affects RNA delivery efficiency, and shorter DMG-PEG is preferred.

[0280] 3. Optimization experiment of LNP component ratio

[0281] The efficiency of LNP containing ionizable lipids in delivering saRNA-GFP-Luc was verified in the 293T cell line. The proportions of each component in the LNP were optimized using the ionizable lipid 17LGluO18U.

[0282] Specific steps:

[0283] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was 17LGluO18U. Ionizable lipids, DSPC, Cholesterol, and DMG-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. Six different molar ratios were used for mixing: Ratio A: Ionizable lipids:Cholesterol:DSPC:DMG-PEG = 40:48:10:2; Ratio B: Ionizable lipids:Cholesterol:DSPC:DMG-PEG = 30:28.5:10:0.75; Ratio C: Ionizable lipids:Cholesterol:DSPC:DMG-PEG = 50:38.5:10:1.5; Ratio D: Ionizable lipids:Cholesterol:DSPC:DMG-PEG = 35:46:16:2.5; Ratio E: Ionizable lipids:Cholesterol:DSPC:DMG-PEG = ... 46.3:42.7:9.4:1.6; the ratio F represents ionizable lipids: Cholesterol:DSPC:DMG-PEG = 46.3:42.7:9.4:1.5.

[0284] (2) Cell transfection efficiency analysis

[0285] Thirty-six hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. The culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are as follows: Figure 2As shown in (b) of the results. The results show that the molar ratio between the components of LNP also affects the RNA delivery efficiency to some extent, and the optimal ratio is F: ionizable lipids: Cholesterol: DSPC: DMG-PEG = 40:48:10:1.5.

[0286] 4. LNP Nitrogen-Phosphorus Ratio Optimization Experiment

[0287] The efficiency of LNP containing ionizable lipids in delivering saRNA-GFP-Luc was validated in the 293T cell line. The nitrogen-phosphorus ratio of the LNP was optimized using the ionizable lipid 17GluO18U.

[0288] Specific steps:

[0289] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was 17GluO18U. Ionizable lipids, DSPC, Cholesterol, and DMG-PEG were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio of ionizable lipid compound 17GluO18U:Cholesterol:DSPC:DMG-PEG = 40:48:10:1.5. The nitrogen-phosphorus ratio of LNP was 4:1, 6:1, 8:1, 12:1, and 16:1, respectively.

[0290] (2) Cell transfection efficiency analysis

[0291] Thirty-six hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. The culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are as follows: Figure 3 As shown in (a) of the results, the transfection efficiency of saRNA-GFP-Luc was optimal when the nitrogen-phosphorus ratio was 6:1.

[0292] 5. Optimization experiment of LNP buffer formulation

[0293] The efficiency of LNP containing ionizable lipids for delivering saRNA-GFP-Luc was validated in the 293T cell line. The buffer formulation of LNP was optimized using the ionizable lipid 17LGluO18U.

[0294] Specific steps:

[0295] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was 17LGluO18U. Ionizable lipids, DSPC, Cholesterol, and DMG-PEG were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio used was 17LGluO18U, 34LGluO18U, or 16AspOC18U:Cholesterol:DSPC:DMG-PEG = 40:48:10:1.5. The premixed RNA solution was sodium acetate or sodium citrate buffer, and the nitrogen-to-phosphorus ratio for preparing LNPs was 6:1.

[0296] (2) Cell transfection efficiency analysis

[0297] Thirty-six hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. The culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are as follows: Figure 3 As shown in (b) above. The results show that when the buffer for preparing nanoparticles is sodium citrate buffer, the RNA delivery efficiency is better than that of sodium acetate buffer. Therefore, sodium citrate buffer is preferred.

[0298] 6. Characterization of modRNA-GFP delivery efficiency

[0299] The efficiency of LNP delivery of modRNA-GFP containing ionizable lipids was validated in the Jurkat T cell line. The transfection efficiency of LNPs was validated using ionizable lipids: 9LGluOC18U, 17LGluO18U, 25DGluO18U, and 29LGluO18U.

[0300] Specific steps:

[0301] (1) Referring to the above experimental method, the Juekat T cell line was used. The ionizable lipids used in the experimental group were: 9LGluOC18U, 17LGluO18U, 25DGluO18U, and 29LGluO18U. The ionizable lipids used in the control group were: SM-10 and ALC-0315. Other components of LNP were DSPC, Cholesterol, and DMG-PEG, which were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL. The ratio of ionizable lipid compounds used was: Cholesterol: DSPC: DMG-PEG = 40:48:10:1.5. The premixed modRNA solution was sodium citrate buffer, and the nitrogen-phosphorus ratio for preparing LNP was 6:1.

[0302] (2) Cell transfection efficiency analysis

[0303] Twenty-four hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. The results are as follows: Figure 4 As shown, the ionizable lipids of the present invention can efficiently deliver nucleic acids to immune cells and are superior to commercial lipids.

[0304] 7. Characterization of in vivo imaging systems

[0305] LNPs containing ionizable lipids 9GluOC18U, 17GluOC18U, 25GluOC18U, 29GluOC18U, 33GluOC18U, 34GluOC18U, and commercial lipids SM-102 and ALC-0315 were used to deliver a chemically synthesized modified messenger ribonucleotide (modRNA-Luc) encoding firefly luciferase in Balb / c mice. Six hours after intravenous injection, the expression of the reporter gene luciferase was detected using an in vivo imaging system (IVIS).

[0306] Specific steps:

[0307] (1) Referring to the above experimental method, the ionizable lipids used in the experimental group were: 9GluOC18U, 17GluOC18U, 25GluOC18U, 29GluOC18U, 33GluOC18U, and 34GluOC18U. The ionizable lipids, DSPC, Cholesterol, and DMG-PEG were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 6 mg / mL, 12 mg / mL, and 5 mg / mL. An appropriate amount of modRNA-Luc was dissolved in sodium acetate buffer (the volume of sodium acetate buffer was twice the total volume of the lipid mixture, pH=4.0). The buffer containing modRNA-Luc was added to the ethanol solution of the lipid mixture and quickly mixed to assemble LNPs. The mixed solution was incubated at room temperature for 15 min. After dialyzing in PBS for 1 h using a dialysis bag (MWCO=14000 MW), a tail vein injection was performed (each injection contained 5 μg of LNP containing modRNA-Luc). The ratio used was ionizable lipids 9GluOC18U, 17GluOC18U, 25GluOC18U, 29GluOC18U, 33GluOC18U, or 34GluOC18U: Cholesterol: DSPC: DMG-PEG = 40:48.5:10:1.5. The premixed RNA solution was sodium citrate buffer, and the nitrogen-to-phosphorus ratio for LNP preparation was 6:1.

[0308] Positive control group: Commercially available lipids ALC-0315 and SM-102 were used to assemble the corresponding positive control LNPs according to published preparation methods. The specific procedures are as follows: ALC-0315 or SM-102, DSPC, Cholesterol, ALC-0159, or DMG-PEG were added. 2000Dissolve the mRNA-Luc in anhydrous ethanol at concentrations of 5 mg / mL, 1.5 mg / mL, 3 mg / mL, and 1 mg / mL respectively, and mix them thoroughly according to the molar ratios of ALC-0315: Cholesterol: DSPC: ALC-0159 = 46.3:42.7:9.4:1.6 or SM-102: Cholesterol:DSPC: DMG-PEG2000 = 50:38.5:10:1.5. Simultaneously, dissolve an appropriate amount of modRNA-Luc in sodium citrate buffer (the volume of sodium citrate buffer should be three times the total volume of the lipid mixture, pH=4.0). Then, rapidly mix the buffer containing mRNA with the lipid mixture solution and incubate at room temperature for 15 min to assemble LNPs. Then, dialyze the LNPs in PBS for 1 h using a dialysis bag (MWCO=14000 MW) and administer a tail vein injection (each injection contains 5 μg of modRNA-Luc LNPs). The nitrogen-phosphorus ratio of ALC-0315, SM-102, and mRNA is 6:1.

[0309] (2) Analysis of in vivo imaging results

[0310] IVIS results 6 hours after tail vein injection (e.g.) Figure 5 , Figure 6 , Figure 7 , Figure 8 The ionizable lipids 29GluOC18U and 34GluOC18U of this invention have better expression intensity than commercial lipids SM-102 and ALC-0315, and have excellent organ targeting ability, enabling precise targeting of the spleen.

[0311] According to literature reports, most ionizable lipid-loaded nucleic acids are expressed in multiple organs throughout the body after intravenous injection, exhibiting hepatophilia. However, the ionizable lipids of this invention can precisely target the spleen. The spleen is the largest immune organ in the body, and LNPs targeting the spleen can bring more significant immunotherapeutic effects in nucleic acid therapy applications.

[0312] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

[0313] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A lipid with an amino acid backbone that can be ionized, characterized in that, It is a compound of formula (A) or formula (B), its pharmaceutically acceptable salt, its stereoisomer or its tautomer; Formula (A); Formula (B); Wherein, R1 is selected from , C2-C10 alkyl, C2-C10 heteroalkyl, aralkyl, or absent; R2 is selected from C3-C24 straight-chain alkyl, C4-C24 straight-chain alkenyl, ... ; n1=2~3, n2=3~24, n3=3~24; X is O or NH; R3 is selected from , , , , , , , , , , , C1-C10 hydroxyalkyl; R6 and R7 are each independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; R4 is selected from C1-C4 hydroxyalkyl; R5 is C1-C4 alkyl, C1-C4 hydroxyalkyl, n4 = 2~3 The "*" indicates a connection point.

2. The amino acid backbone ionizable lipid according to claim 1, characterized in that, The structure of the ionizable lipid with the amino acid backbone is shown in any of the structural formulas (1) to (104): Equation (1) Equation (2); Equation (3) Equation (4); Equation (5) Equation (6); Equation (7) Equation (8); Equation (9) Equation (10); Equation (11) Equation (12); Equation (13) Equation (14); Equation (15) Equation (16); Equation (17) Equation (18); Equation (19) Equation (20); Equation (21) Equation (22); Equation (23) Equation (24); Equation (25) Equation (26); Equation (27); Equation (28); Equation (29); Equation (30); Equation (31) Equation (32); Equation (33) Equation (34); Equation (35) Equation (36); Equation (37) Equation (38); Equation (39) Equation (40); Equation (41) Equation (42); Equation (43) Equation (44); Equation (45) Equation (46); Equation (47) Equation (48); Equation (49) Equation (50); Equation (51) Equation (52); Equation (53) Equation (54); Equation (55) Equation (56); Equation (57) Equation (58); Equation (59) Equation (60); Equation (61); Equation (62); Equation (63); Equation (64); Equation (65) Equation (66); Equation (67) Equation (68); Equation (69) Equation (70); Equation (71) Equation (72); Equation (73) Equation (74); Equation (75) Equation (76); Equation (77) Equation (78); Equation (79) Equation (80); Equation (81) Equation (82); Equation (83) Equation (84); Equation (85) Equation (86); Equation (87) Equation (88); Equation (89) Equation (90); Equation (91) Equation (92); Equation (93) Equation (94); Equation (95) Equation (96); Equation (97) Equation (98); Equation (99) Equation (100); Equation (101) Equation (102); Equation (103) Equation (104).

3. The method for preparing the amino acid backbone ionizable lipid according to claim 1 or 2, characterized in that, The process includes the following steps: reacting a tail compound containing an amino acid structure or its stereoisomers or tautomers with an organic amine compound via a Michael addition reaction to obtain an ionizable lipid with the amino acid skeleton; The structural formula of the tail compound containing the amino acid structure is shown in formula (a): Formula (a); R1, R2, and X are as described in claim 1.

4. The method for preparing an ionizable lipid with an amino acid backbone according to claim 3, characterized in that, The organic amine compound is selected from one of the following compounds: 。 5. The method for preparing an ionizable lipid with an amino acid backbone according to claim 3, characterized in that, The preparation method of the tail compound containing the amino acid structure includes the following steps: reacting acryloyl chloride with compound a1 Tail compounds containing amino acid structures were prepared by esterification.

6. A lipid composition, characterized in that, Includes the amino acid backbone of claim 1 or 2, which can ionize lipids, sterols, and polyethylene glycol-modified lipids.

7. The use of the amino acid backbone ionizable lipid of claim 1 or 2, or the lipid composition of claim 6, in the preparation of a bioactive substance delivery system, characterized in that, The delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle, or microbubble.

8. A pharmaceutical composition, characterized in that, It includes the lipid composition and active substance of claim 6; the active substance includes at least one of nucleic acid molecules, small molecule compounds, polypeptides, and proteins.

9. The pharmaceutical composition according to claim 8, characterized in that, When the active substance includes nucleic acid molecules, the nitrogen-to-phosphorus ratio of the ionizable lipid to the nucleic acid molecule in the amino acid backbone is (1~100):

1.

10. The use of the amino acid backbone ionizable lipid of claim 1 or 2, or the pharmaceutical composition of claim 8 or 9, in the preparation of nucleic acid drugs, gene vaccines, polypeptide or protein drugs, or small molecule drugs.

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